A recombinant protein is a protein obtained by applying recombinant DNA or RNA technologies. Various recombinant proteins widely produced by in vitro protein expression technologies can be used in the fields of medicine, food, daily chemical, etc.
E. coli
Escherichia coli (E. coli) has a clear genetic background, which has various vectors and hosts. It is not only the most commonly used protein expression system but also the most economical one. By optimizing the expression system, such as the compatibility test of the expression vector and the expression strain, the optimization of the induced expression conditions, and the renaturation, the problems of protein expression can be solved to a great extent. This method applies to the proteins that do not require high glycosylation function.
Advantages:
* The genetic background of E. coli is clear, which has fast reproduction and strong anti-pollution ability.
* High expression level, relatively simple separation and purification of expression products, and good stability
* The types of commercial vectors and strains are satisfactorily widespread.
Disadvantages:
* E. coli does not have the function of eukaryotic post-transcriptional processing, and cannot perform mRNA splicing, so it can only express cDNA but not eukaryotic genomic genes.
* E. coli does not have the function of eukaryotic post-translational processing. The resulting protein cannot be modified by glycosylation or phosphorylation, and it is difficult to form correct disulfide bond pairing and spatial conformation folding.
* The protein expressed by E. coli is often insoluble and will aggregate into inclusion bodies in the bacteria, especially when the amount of expressed target protein exceeds 10% of the total bacterial body protein.
* E. coli may produce some pyrogenic sources (endotoxin), which itself contains endotoxin and toxic protein. Endotoxin needs to be removed in the purification of the target protein, which also limits the application.
Saccharomyces cerevisiae
The expression of recombinant proteins with Saccharomyces cerevisiae started in 1981. Saccharomyces cerevisiae is suitable for expressing scientifically and commercially valuable heterologous proteins. Because it does not produce toxins, and the wide application in food production, it has become the best host bacteria for pharmaceutical protein production. A series of insulins produced by yeast that are widely used in the treatment of diabetes worldwide, which is a crucial biotechnological application.
Advantages:
* It has a high expression level, a good choice for secreted protein or cell expression.
* Low cost with simple culture conditions
* Has post-translational modifications of most eukaryotes, effective protein folding, and no endotoxin.
Disadvantages:
The expression level is lower than that of Pichia pastoris, and its secretion capacity may be lower than that of Pichia pastoris. The structure of the over-glycosylated N-terminal glycosyl chain is sensitizing.
Methanol Yeast
Methanol yeast uses methanol as the sole carbon source. H. Polymorpha, Candida Bodinii, and Pichia Pastoris are the major sources, among which Pichia Pastoris is the most widely applied gene expression system. It has unmatched high expression characteristics and has been regarded as one of the most promising tools for protein production.
Advantages:
* It has a high expression level within a short production cycle, which is a good choice for secreted protein or intracellular expression.
* It has strong protein secretion ability and the products can be used for simple purification and extensive post-translational modification. It performs better in the N-terminal glycosylation than Saccharomyces cerevisiae.
Disadvantages:
There are certain hazards of using methanol as an inducer, and its glycosylation is still different from mammalian cells.
Baculovirus-insect Cell
Baculovirus-insect cell expression system uses insect baculovirus as the foreign gene carrier and insect and insect cells as receptors. Insect cells and mammalian cells have similar translation and post-translational protein modification modes and capabilities, including glycosylation, phosphorylation, acylation, signal peptide excision, and peptide cleavage and decomposition. The biological activity of the resulting recombinant protein, such as antigenicity, immunogenicity, and function, is similar to that of natural protein. This system has received increasing attention and has been widely used.
Mammalian cells can be used to express exogenous recombinant protein plasmid transfection and viral vector infection. It takes several weeks or even months to obtain stable transfected cells by plasmid transfection, while cells can be quickly infected through the viral expression system, and the foreign gene can be integrated into the viral vector within a few days. Mammalian cell expression vectors must contain control elements such as prokaryotic sequences, promoters, enhancers, selectable marker genes, terminator, and polynucleotide signals.
Animal & Plant Cell Reactors
Animal and plant bioreactors are genetic engineering methods that use common animals or crops as "biological factories" to produce biological agents with high economic added value such as medical protein, industrial and agricultural enzymes, special carbohydrates, biodegradable plastics, lipids, and other secondary metabolites through large-scale cultivation and planting. Animals and plants can express proteins from animals, bacteria, viruses, and plants themselves, which are easy to cultivate and produce on a large scale, and have special advantages in gene expression, modification, and safety. Therefore, the use of animals and plants to produce exogenous proteins has great prospects.
In the coming years, the improvement of biotech drugs, new products, new formulations, and the like is promising, with more and more functional proteins with large molecular weight and complex structures developed into biotechnology drugs, and therapeutic antibodies becoming the second innovation climax in the field of biopharmaceuticals. In order to improve drug performance, reorganization, fusion proteinization, and in vitro chemical modification will receive increasing attention.